G. Engler

Dominant negative mutants of the Cdc2 kinase uncouple cell division from iterative plant development.

Because plant cells do not move and are surrounded by a rigid cell wall, cell division rates and patterns are believed to be directly responsible for generating new structures throughout development. To study the relationship between cell division and morphogenesis, transgenic tobacco and Arabidopsis plants were constructed expressing dominant mutations in a key regulator of the Arabidopsis cell cycle, the Cdc2a kinase. Plants constitutively overproducing the wild‐type Cdc2a or the mutant form predicted to accelerate the cell cycle did not exhibit a significantly altered development. In contrast, a mutation expected to arrest the cell cycle abolished cell division when expressed in Arabidopsis, whereas some tobacco plants constitutively producing this mutant protein were recovered. These plants had a reduced histone H1 kinase activity and contained considerably fewer cells. These cells were, however, much larger and underwent normal differentiation. Morphogenesis, histogenesis and developmental timing were unaffected. The results indicate that, in plants, the developmental controls defining shape can act independently from cell division rates.

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called ‘opines’, e. g. octopine (N-α-(D-l-carboxyethyl)-L-arginine) and nopaline (N-α-(l, 3-dicarboxypropyl)-L-arginine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a ‘genetic colonization’ of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.

Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens.

A detailed physical map of the homologous and non-homologous regions between an octopine (pTiAch5) and a nopaline (pTiC58) Ti plasmid was determined by Southern type hybridization and by electron microscope heteroduplex analysis. This map was correlated with the functional maps of both plasmids. For the Southern type hybridizations, total labelled pTiAch5 DNA was hybridized to Southern blots of restriction fragments from a series of hybrid plasmids containing overlapping segments of the whole TiC58 plasmid. Reciprocal experiments were also carried out. The common sequences between the two plasmids (±30%) are restricted to four major stretches of homology. Analysis of heteroduplexes between pTiAch5 and several hybrid plasmids containing specific regions of pTiC58, and of heteroduplexes between hybrid plasmids derived from pTiC58 and pTiAch5 provided a detailed map of the fine structure of the four major homology regions. Two regions are distributed in the same relative order as compared to a common reference point, and two are inversed. Three regions contain a number of small, mostly asymmetrical substitution loops. Several regions distributed over the common DNA sequences were found to be partially homologous.

Distribution of the rDNA and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana

The distribution of the ribosomal RNA (rRNA) genes and three classes of highly repetitive DNA in the chromatin of interphase nuclei of Arabidopsis thaliana was studied for the first time through non-isotopic in situ hybridization and luminescence digital imaging microscopy. Each of the three classes of highly repetitive DNA exhibited a characteristic hybridization pattern, and one class was seen to be primarily localized on two chromocentres, which would allow it to distinguish a particular chromosome. The rDNA was consistently localized on the two largest chromocentres and on one or two smaller chromocentres. A limited number of nuclei exhibited more than four labelled chromocentres, indicative of either polypoidy or differential amplification of the rDNA. In nuclei where the nucleolus could be clearly observed, the nucleolar associated chromocentres (NACs) were seen to be labelled by the ribosomal DNA (rDNA) probe.

Spontaneous formation of cointegrates of the oncogenic Ti-plasmid and the wide-host-range P-plasmid RP4.

A pTiB6S3::RP4 cointegrate and a pTiC58::RP4 cointegrate plasmid were isolated. They are formed by the covalent linkage of the respective pTiB6S3 and pTiC58 plasmids of Agrobacterium tumefaciens and of the RP4 factor. They carry the genetic markers of both parental molecules, and these markers are transferred as a unit under the same circumstances as the RP4 factor alone. The pTiB6S3::RP4 plasmid retains its oncogenic capacity and does not dissociate with a detectable frequency in Agrobacterium strains. The pTiC58::RP4 cointegrate readily dissociates apparently with exact separation of the genetic markers of pTiC58 and of RP4. The cointegrate pTiC58::RP4 state is non-oncogenic whereas after dissociation the oncogenicity of pTiC58 is restored.

The Interaction of Agrobacterium Ti-Plasmid DNA and Plant-Cells

The tumour-inducing plasmids of Agrobacterium tumefaciens (Ti-plasmids) reveal several interesting properties. They are catabolic plasmids, which, instead of rendering Agrobacterium strains capable of catabolizing compounds found in Nature, force a plant to synthesize these catabolites (denoted ‘opines’). This situation is obtained by insertion of a segment of the Ti-plasmid (the T-DNA) into the plant nucleus, where T-DNA genes become expressed and intervene in the biosynthesis of these opines. Cells containing the T-DNA behave as neoplasms (crown gall cells). Southern blotting shows that the insertion process responsible for T-DNA transfer probably recognizes special sequences on the T-DNA since the length of the T-DNA segment observed in different, independently isolated tumour lines was found to be similar. For the nopaline Tiplasmids both left-hand and right-hand borders were found to be constant. For the octopine plasmid the left border was constant and at least two classes of right-hand borders were found. Upon redifferentiation of the transformed plant cells, the T-DNA was found to be conserved in all somatic cells examined. However, small deletions at the border fragments of the T-DNA have been observed. The exact arrangement and copy number of the T-DNA in a nucleus is still under study, but genomic cloning has already revealed that an interspersed tandem arrangement is dominant in nopaline tumours. Clones containing both the right border of one T-DNA and the left border of the neighbouring tandem T-DNA were isolated. In order to identify the different T-plasmid encoded functions an extensive use was made of transposon insertion mutagenesis. When an antibiotic resistance transposon was inserted into the non-essential regions of the T-DNA, a linked transfer to the plant DNA of the transposon together with the T-DNA was observed. This indicates that Ti-plasmids are possible vectors for genetic engineering in plants. A strategy is described for insertion of any cloned DNA segment into the T-DNA.

The nopaline C58 T-DNA region is transcribed in Agrobacterium tumefaciens

SummaryWe have analyzed the transcription of the T-DNA region of the nopaline Ti plasmid C58 in Agrobacterium tumefaciens. This T-DNA region is usually expressed in plant cells following transformation with Agrobacterium. However, we show that there are also at least twelve distinct transcrips present in Agrobacterium with significant differences in their steady-state levels. To analyze the bacterial transcription of the nopaline T-DNA region, cDNA prepared from total bacterial RNA was hybridized to different nitrocellulose-bound subfragments of the T-DNA region. The direction of transcription of the different T-DNA region-encoded bacterial transcripts was determined by hybridizing labeled total RNA to both complementary strands of subfragments of the T-DNA region. Electron microscopic R-loop mapping was used to show that the promoter sequences of the T-DNA region, which are utilized by Agrobacterium tumefaciens, are also recognized by the E. coli RNA polymerase in vitro. Of the twelve T-DNA region transcripts found in Agrobacterium, at least seven may be the same as those found in plant cells.

Plasmid Mobilization as a Tool for in Vivo Genetic Engineering

Mutagenesis through the insertion of transposons has proved to be an invaluable technique for mapping the genes of complex plasmids1. No selection for a mutant phenotype has to be devised, but a straightforward selection for the antibiotic resistance markers, encoded by the transposon, is sufficient to identify the presence of a mutant plasmid.

Vitellogenin knockdown strongly affects cotton boll weevil egg viability but not the number of eggs laid by females

Vitellogenin (Vg), a yolk protein precursor, is the primary egg nutrient source involved in insect reproduction and embryo development. The Cotton Boll weevil (CBW) Anthonomus grandis Boheman, the most important cotton pest in Americas, accumulates large amounts of Vg during reproduction. However, the precise role of this protein during embryo development in this insect remains unknown. Herein, we investigated the effects of vitellogenin (AgraVg) knockdown on the egg-laying and egg viability in A. grandis females, and also characterized morphologically the unviable eggs. AgraVg transcripts were found during all developmental stages of A. grandis, with highest abundance in females. Silencing of AgraVg culminated in a significant reduction in transcript amount, around 90%. Despite this transcriptional reduction, egg-laying was not affected in dsRNA-treated females but almost 100% of the eggs lost their viability. Eggs from dsRNA-treated females showed aberrant embryos phenotype suggesting interference at different stages of embryonic development. Unlike for other insects, the AgraVg knockdown did not affect the egg-laying ability of A. grandis, but hampered A. grandis reproduction by perturbing embryo development. We concluded that the Vg protein is essential for A. grandis reproduction and a good candidate to bio-engineer the resistance against this devastating cotton pest.